Browsing by Subject "antibacterial"

The emergence of resistance to existing antibiotic drugs necessitates the development of new strategies to treat bacterial infections. Copper (Cu) has been used since ancient times to inhibit bacterial growth and has recently experienced a resurgence in its clinical utility as an antimicrobial coating for surfaces in hospitals. Small molecule chelators that bind Cu have also been shown to have antibacterial activity and are believed to disrupt metal homeostasis within the microbes they kill. Molecules called ionophores shuttle Cu into the cell to poison it. However, the antibacterial modes of action behind Cu and small molecule ionophores are not well understood. In this work, we employ a variety of biological, spectrometric, and proteomic techniques to study how Cu and a small molecule ionophore called pyrithione (PT) kill bacteria. First, we present antibacterial susceptibility assays that demonstrate PT and a β-lactamase-activated prodrug of PT called PcephPT kill bacteria in a Cu-dependent manner. Cu hyperaccumulated in cells that were cotreated with low-micromolar Cu and either PT or PcephPT, demonstrating their activity as metal-shuttling ionophores. Next, proteome-wide protein expression level and stability measurements were used to probe treatment-induced cellular changes after E. coli were exposed to Cu in the absence and presence of PT or PcephPT. The stability-based study identified key protein targets such as the metabolic enzymes glyceraldehyde-3-phosphate dehydrogenase and isocitrate dehydrogenase, whose activities were confirmed to be inhibited by PT-induced copper toxicity in enzymatic assays. Finally, the impact of Cu on the proteome was further investigated in a metal-induced protein precipitation experiment. Unlike other divalent first row transition metals, low millimolar Cu induced complete protein precipitation from E. coli lysate. Protein solubility was restored by addition of Cu chelators, showing that Cu-induced protein precipitation is reversible. We then obtained Cu precipitation curves for over 800 proteins and saw that some were more sensitive while others were more tolerant to precipitation by Cu. Finally, we analyzed the data set to better understand what biophysical characteristics of the proteins may contribute to making them sensitive or tolerant to precipitation by Cu.

!Drug discovery typically involves structure-based drug design based on three-dimensional protein structures and hit/lead compound identification and optimization. Herein, this technique was used to overcome several obstacles associated with the developing of antibiotics, anticancer agents, and antifungals and reveal critical insights into the corresponding structure-activity relationships (SARs).Phospho-N-acetyl-muramyl-pentapeptide translocase (MraY) is an important membrane enzyme involved in the early-stage biosynthesis of bacterial peptidoglycans. As the inhibition of MraY leads to bacterial cell lysis, such MraY inhibitors (e.g., muraymycin) hold great promise for antibiotic development. However, the structural complexity of muraymycin makes its synthesis and practical applications challenging. Hence, we synthesized several muraymycin analogs with reduced structural complexity and better synthetic tractability and identified the moieties responsible for their biological activity to facilitate the development of muraymycin-derived antibiotics. Translesion synthesis (TLS) is a major mechanism that enables bypass replication over DNA lesions and promotes the formation of mutagenic DNA. Rev1/Pol ζ–mediated TLS plays an important role in cisplatin-induced mutations, and thus, the Rev1/Pol ζ interface is an attractive target for small-molecule TLS inhibitors. Herein, we aimed to develop TLS inhibitors as potential anticancer agents based on the recently reported inhibitor of the Rev1-Rev7 interaction, JH-RE-06. Despite its high potency, JH-RE-06 is poorly soluble in aqueous media and is therefore a limitation for further development. To overcome this limitation and identify novel anticancer agents, we prepared various JH-RE-06 analogs and studied the related SARs, to determine the critical functional groups for improving the biological activity improvement and aqueous solubility. Currently, fungal infections, which are particularly dangerous to immunocompromised patients, are a frequent cause of a death. However, the similarities between the eukaryotic physiologies of fungal pathogens and their hosts render targeting of the pathogen without causing side effects in the host challenging. Calcineurin (CN) plays a major role in invasive fungal diseases and is therefore a promising target for antifungal drug development. FK506, which is an approved CN inhibitor, exhibits promising activity but an insufficient selectivity because of its strong immunosuppressive effect. Therefore, in developing antifungal agents, we exploited the major structural differences between the CN-FK506-FKBP12 ternary complexes of humans and fungi and developed FK506/520 analogs targeting these complexes. The synthesized analogs retained the parent antifungal efficacy while exhibiting lower immunosuppressive activities and improved therapeutic efficacies both in vivo and in vitro.

Antimicrobial peptides (AMPs) are part of the innate immune system that is widely distributed in nature, acting as a defense mechanism against invading microorganisms. AMPs have potent antimicrobial activity against a range of microorganisms including fungi, bacteria and viruses. In view of growing multidrug resistance, AMPs are increasingly being viewed as potential therapeutic agents with a novel mechanism of action. Mastoparan is a natural, highly positively charged AMP derived from the venom of wasps. It was originally of interest based on its inherent mast cell degranulation activity. Previously, mastoparan has been shown to exhibit antimicrobial activity in vitro however these studies have been limited in scope. Here we hypothesize that mastoparan possess the capacity to be a potent broad spectrum antibacterial agent including activity against multidrug resistant bacteria.

We examined the scope of antibacterial activity exhibited by mastoparan using a variety of antimicrobial susceptibility tests and have utilized a bacterial skin infection (S. aureus) model to determine the potential of mastoparan to serve as a therapeutic agent. We tested mastoparan against 4 Gram-positive clinical isolates (e.g., S. aureus, and E. faecium), 9 Gram-negative clinical isolates (e.g., E. coli, P. aeruginosa, and B. cepacia), and 4 multidrug resistant clinical isolates (e.g., MRSA, ESBL E.coli, and ESBL K. pneumonia). These studies reveal that mastoparan exhibits broad spectrum activity against both Gram-negative (MIC: 1.9 - 125 &mug/ml) and Gram-positive (MIC: 15.6 - 125 &mug/ml) bacteria and against multidrug resistant bacteria (MIC: 7.8 - 125 &mug/ml). We also demonstrated that mastoparan disrupts the bacterial membrane, exhibits fast acting antibacterial activity and is highly effective against both multiplying and non-multiplying bacteria. Furthermore, we have shown that mastoparan demonstrates efficacy as a topical antimicrobial agent reducing lesion size by up to 79% and the amount of bacteria recovered from skin lesions by up to a 98% reduction. Based on these results we conclude that mastoparan is a highly effective antibacterial agent and is therefore a potential alternative to currently antibiotics. Mastoparan offers a promising new therapeutic option for treating bacterial infections.